Cell and tissue culture techniques are becoming increasingly more important to basic and applied life science research. The development of culture containers and cell attachment substrates have been driven by the need to produce an environment that resembles the in vivo state as closely as possible. As a result, permeable supports including microporous membranes in a variety of configurations and devices, have become the standard method for the culture of polarized cells. Significant improvements in experimental design have resulted because permeable membrane filters permit cells to feed basolaterally and thereby carry out metabolic activities in a more natural fashion.
Membrane filters have been used as cell growth substrates since the 1950's and the transfilter mesonephric induction studies of Grobstein. Adapted over the years to a variety of cell types and numerous applications, porous membrane filters are now recognized as providing significant advantages over solid, impermeable cell growth substrates. For polarized structures such as epithelial cells, the use of permeable supports allows cells to be studied under more natural conditions. Cellular differentiation proceeds to higher levels resulting in cells that morphologically and functionally better represent their in vivo counterparts.
Cellular activities such as transport, absorption and secretion can also be studied under more natural conditions since cells grown on filters provide convenient, independent access to apical and basolateral plasma membrane domains.
The use of permeable support systems for cell culture has proven to be a valuable tool in the cell biology laboratory.
In prior application Ser. No. 06/841,562 supra, one commercially successful system for growing tissue cultures is shown. Individual filter wells are shown as used in the wells of multi-well cluster plates. That system, however, is not suitable for use with the extensive amount of auxiliary equipment developed to handle multi-well cluster plates in the standardized 96-well format.
Industry standard microtest cluster plates are laid out with 96 wells in an 8.times.12 matrix (mutually perpendicular 8 and 12 well rows) with a spacing of 0.355 inches between the center lines of rows both in the x and y directions. In addition, the height, length and width dimensions of the microtest 96-well plates are standardized. This standardization has resulted in the development of a large amount of auxiliary equipment being developed specifically for the 96-well formats. The equipment includes devices that load and unload precise volumes of liquid in multiples of either 8, 12, or 96 wells at a time. In addition, equipment is available to transmit light through the individual wells and to read colorimetric changes or chemiluminescence in individual wells (as a result of tests performed in each well). Some of this equipment is automated and instrumented to record, analyze and manipulate the data recorded.
The present invention includes multi-well plates having wells of substantially greater diameter and volume than the wells in the standardized 96-well plates but nevertheless are capable of being used with the equipment designed for the 96-well format. More particularly, the present invention includes a 48-well plate wherein the length, width and height of the plate is identical to the industry standard established for 96-well plates. The well diameters of the 48-well plate are maximized by staggering the wells in adjacent rows and the coincidence of the walls of adjacent wells at their points of tangency.
The present invention also includes filter plates composed of a single filter well or an array of 4, 8, 24 or 48 wells whose formats conform to the format of the multi-well cluster plate of this invention and sized so that they may extend into the wells (reservoir wells) of the cluster plate. The filter plates include means for positioning the filter wells in selected positions within the wells of the cluster plate so as to minimize capillary action between the filter and reservoir well walls and to space the filters well a preselected distance above the bottom walls of the reservoir wells. The filter plates also include means to provide access to the interior of the filter wells and the lower portion of the reservoir wells so that liquid may be introduced in precise quantities to those regions. In accordance with one embodiment of this invention, individual filter wells may be broken away from the others in the filter plate for separate analysis.
The system of this invention also includes a common reservoir that receives all of the filter wells of the filter plate so that tissue culture monolayers may be grown in multiples prior to use of the filter plate with the multi-well cluster plate of the present invention for analysis on 96-well format auxiliary equipment.
In accordance with the present invention, the 48 wells of the multi-well plate are arranged in 12 rows of 4 wells each, and the center lines of the 12 rows are spaced apart the same distance, as the spacing between the rows of wells in the standard 96-well plate, that is, 0.355 inches. The centers of the four wells in each row are spaced apart twice that distance, namely, 0.710 inches, and the wells in adjacent rows are offset from one another so that the centers of the wells in a particular row are aligned with the midpoint between the centers of adjacent wells in the adjacent rows. In this fashion, the centers of the wells in alternate rows align with the odd and even-numbered wells of corresponding rows in the 96-well format. The arrangement of the wells allows their diameters to be maximized to approximately 1.5 times the well diameter in the standard 96-well cluster plates. The large wells of the plate in turn maximize the clearance with the walls of the filter wells in the system so as to avoid capillary action. The system also enables 96-well ELISA test readers to be used in combination with multiple filter wells for applications such as chemotaxis studies, migration assays, cell uptake analysis, diagnostic tests where membranes are used for separation, sorting, affinity binding, etc. The contents of the entire filter well and reservoir well, may be read depending upon the requirement of the assay.
The present invention will be better understood and appreciated from the following detailed description read in connection with the accompanying drawings in which: